Glucagon-like Peptide-1 Receptor Agonists in Patients with Type 2 Diabetes Mellitus and Nonalcoholic Fatty Liver Disease—Current Background, Hopes, and Perspectives

Nonalcoholic fatty liver disease (NAFLD) represents the most common chronic liver disease worldwide, reaching one of the highest prevalences in patients with type 2 diabetes mellitus (T2DM). For now, no specific pharmacologic therapies are approved to prevent or treat NAFLD. Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are currently evaluated as potential candidates for NAFLD treatment in patients with T2DM. Some representatives of this class of antihyperglycemic agents emerged as potentially beneficial in patients with NAFLD after several research studies suggested they reduce hepatic steatosis, ameliorate lesions of nonalcoholic steatohepatitis (NASH), or delay the progression of fibrosis in this population. The aim of this review is to summarize the body of evidence supporting the effectiveness of GLP-1RA therapy in the management of T2DM complicated with NAFLD, describing the studies that evaluated the effects of these glucose-lowering agents in fatty liver disease and fibrosis, their possible mechanistic justification, current evidence-based recommendations, and the next steps to be developed in the field of pharmacological innovation.


Introduction
Type 2 diabetes mellitus (T2DM) is a chronic health condition that shares a wide range of risk factors and pathogenic elements with obesity and nonalcoholic fatty liver disease (NAFLD) [1]. The prevalence of diabetes in adults aged 20-79 years worldwide is estimated at 10.5%, of whom more than 90% have T2DM [2].
Along with the diabetes pandemic, today, NAFLD represents the most prevalent liver disease worldwide and is perceived as the hepatic manifestation of the metabolic syndrome [3]. The global prevalence of NAFLD among adults is currently 25-30% [4] and is predicted to reach 55.7% in 2040 [5]. More than 90% of the patients with severe obesity, and 50% of T2DM patients have NAFLD [4,6]. T2DM is often associated with multiple organ dysfunction, including eye, kidney, liver, and cardiovascular disease. NAFLD represents the least screened diabetes mellitus complication today; this is a discouraging finding, given the rapid progression towards nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, or even hepatocellular carcinoma, all leading to a rising liver-related mortality [7].
The relationship between NAFLD and T2DM is bidirectional and requires increased awareness [8]. The presence of diabetes promotes the progression of NAFLD toward

Basic Concepts in the Management of Patients with T2DM and NAFLD
Screening of people at risk for liver disease is advised for all patients with T2DM, as well as in individuals who are overweight or living with obesity, in people with metabolic syndrome, or in those living with cardiovascular disease [14,15].
Once the diagnosis of NAFLD is established, an intensive management strategy needs to be implemented. The cornerstone of NAFLD treatment remains lifestyle optimization; paradoxically enough, given the increased incidence of the disease and the vital risks involved by its progression, there are still no drugs specifically approved for NAFLD therapy [14,16]. The treatment objectives involve avoidance or delayed progression of diabetes-related complications (cardiovascular disease and liver cancer included) by improving the metabolic profile, and the reduction of the liver fat load, inflammation, or even fibrosis, given the liver's ability to regenerate [14].
Lifestyle modification in the management of NAFLD includes initiation and maintenance of weight loss through nutritional intervention and increased physical activity patterns [17,18]. Nevertheless, nonpharmacological therapy displays limited efficacy for weight-related outcomes, which impairs its ability to change the course of liver disease [19].
Antihyperglycemic agents supporting weight loss in patients with T2DM have shown beneficial effects in NAFLD [20]. However, it appears that improvement of glycemic control in patients with co-existent NAFLD and diabetes may be associated with an improvement in NAFLD independently of the lost weight [21]. Glycemic outcomes cannot be considered outside the need to tackle metabolic traits such as dyslipidemia or obesity, which improves the liver condition in patients with or without diabetes [22].
Along with sodium-glucose cotransporter 2 (SGLT2) inhibitors, GLP-1RAs have become the first-line therapy for T2DM due to their potent glucose-lowering effects and their ability to reduce the cardiovascular risk irrespective of the glycemic improvement or the weight loss [23].
GLP-1RAs belong to the incretin-based therapies with subcutaneous or oral administration that are currently approved for the treatment of T2DM and obesity (Table 1) [24,25]. They are intensively scrutinized, given their beneficial impact on the glycemic control, their ability to reduce the visceral adipose tissue and the total body weight, and their cardioreno-metabolic benefits in patients with T2DM and obesity ( Figure 1) [26][27][28]. Therefore, the inference of their benefits in NAFLD-complicated diabetes, or even in NAFLD without associated diabetes, was only a step further [29]. Some data even suggest that GLP-1RAs have metabolic effects related to NAFLD pathophysiology [22]. Some of the high-impact guidelines for NAFLD have already embedded the use of GLP-1RAs (Table 2). United States guidelines were the first to support the use of GLP-1RAs in patients with T2DM and NASH [12,15]. As of now, further studies are still needed to clarify the role of GLP-1RAs as an NAFLD-targeted therapy in persons with or without diabetes, including their ability to interfere with each of the histological changes seen in this metabolic liver disease, as a valuable addition to their well-recognized capacity to improve cardiometabolic health [39].
to clarify the role of GLP-1RAs as an NAFLD-targeted therapy in persons with or without diabetes, including their ability to interfere with each of the histological changes seen in this metabolic liver disease, as a valuable addition to their well-recognized capacity to improve cardiometabolic health [39].  Recommend pioglitazone and GLP-1RAs for people with T2DM and biopsy-proved NASH.
Consider treating T2DM with GLP-1RAs or pioglitazone in the situation of possible NASH with modified noninvasive tests and elevated levels of hepatic enzymes, also offering cardiometabolic benefits, even in pediatric obesity and T2DM.
APASL clinical practice guidelines for the diagnosis and management of metabolicassociated fatty liver disease, 2020 [40] Insufficient evidence in the Asian population.

EASL-EASD-EASO Clinical
Practice Guidelines for the management of nonalcoholic fatty liver disease, 2016 [17] Pharmacotherapy reserved for patients with NASH and significant fibrosis (≥stage F2) or NASH with a high risk for disease progression (elevated ALT, T2DM, MS   Recommend pioglitazone and GLP-1RAs for people with T2DM and biopsy-proved NASH.
Consider treating T2DM with GLP-1RAs or pioglitazone in the situation of possible NASH with modified noninvasive tests and elevated levels of hepatic enzymes, also offering cardiometabolic benefits, even in pediatric obesity and T2DM.
APASL clinical practice guidelines for the diagnosis and management of metabolic-associated fatty liver disease, 2020 [40] Insufficient evidence in the Asian population.
EASL-EASD-EASO Clinical Practice Guidelines for the management of nonalcoholic fatty liver disease, 2016 [17] Pharmacotherapy reserved for patients with NASH and significant fibrosis (≥stage F2) or NASH with a high risk for disease progression (elevated ALT, T2DM, MS
Clinical trials, randomized controlled trials (RCT), and multicenter studies examining the effectiveness of GLP-1RA therapy on NAFLD in patients with T2DM were included.
Studies that did not include subjects with T2DM were excluded. Only articles available in English and in full text were considered. To avoid omissions, other types of articles such as reviews, guidelines, systematic reviews, and meta-analyses were consulted. We also carefully examined the reference lists of the previously selected articles for other eligible and relevant articles. All the relevant information derived from the selected articles has been compiled into text or table form.
Relevant data were extracted by two independent authors who selected information related to the study characteristics (author, year of publication, country of origin, sample size, follow-up duration), the therapeutic regimen, diagnostic methods, and treatment outcomes (changes in liver biomarkers, steatosis and fibrosis indices, liver fat content, and histological modifications). An independent third author performed blinded checking of the data and disparities were resolved through consensus. Table 3 summarizes all data (clinical trials, RCT, multicenter studies, post hoc analyses) with at least a 12-week follow-up collected from patients with T2DM and NAFLD based on the use of exenatide, liraglutide, dulaglutide, and semaglutide in various doses compared with either placebo or other glucose-lowering agents, regardless of the population sample, primary endpoints, or investigation methods. No studies with efpeglenatide, lixisenatide, or albiglutide were found. All cited references summarized results of human studies, with both male and female adult patients included. Patients in various studies had different degrees of NAFLD, from steatosis to NASH or fibrosis. A total of 32 studies were found, including eight studies with exenatide, sixteen with liraglutide, six studies with semaglutide, and two studies with dulaglutide, all with positive results of improving NAFLD. Among all, liraglutide was by far the most investigated GLP-1RA for the management of NAFLD in patients with T2DM. In addition to the effects of ameliorating liver biomarkers, steatosis and fibrosis indices, liver fat content, and histological data, some studies also correlated the amounts of visceral adipose tissue and subcutaneous adipose tissue to NAFLD, and found improvements after the treatment with GLP-1RAs was implemented.

Changes in Serum Liver Enzymes
Exenatide was the first GLP-1RA to show, besides its favorable effects on HbA1c, insulin resistance, body weight, and blood pressure, an improvement of the hepatic enzymes in patients with T2DM [45,49]. Nowadays, exenatide is less used because of the lack of cardiorenal benefits [74] that were later demonstrated by the new generation of long-acting GLP-1RAs, such as liraglutide, dulaglutide, and semaglutide [75].
Liraglutide appears to be, for now, the most studied GLP-1RA in NAFLD, raising high hopes for its ability to prevent and treat the metabolic liver disease [76,77]. A metaanalysis of the LEAD program that included 4442 patients with T2DM assessed the effects of liraglutide on liver parameters after a 26-week therapy [78]. Liraglutide 1.8 mg per day improved liver enzymes, while smaller doses did not show significant effects after adjustments to body weight were applied [78].
The findings of a post hoc analysis of 1499 subjects from the AWARD trials with dulaglutide also showed a decrease in serum aminotransferases vs. placebo (−8.8 IU/L vs. −6.7 IU/L) in patients with T2DM and NASH after the administration of dulaglutide 1.5 mg per week [79].
Semaglutide is another GLP-1RA that has also gained interest in the treatment of steatohepatitis, as several studies in patients with T2DM and obesity reported beneficial changes in liver enzymes and inflammatory biomarkers such as high-sensitivity C-reactive protein [80,81].

Effects on Composite Indices of Hepatic Steatosis and Fibrosis
Improvement of noninvasive scores for NAFLD was observed in a trial by Gastaldelli et al., which assessed the efficacy of exenatide once weekly in association with dapagliflozin in T2DM patients. Fatty liver index (FLI), NAFLD liver fat score (NLFS), Fibrosis-4 (FIB-4) index, and NAFLD fibrosis score (NFS) decreased under the synergic effects of exenatide and dapagliflozin [42]. On the other hand, multiple glucose-lowering agents were able to improve NAFLD scores in patients with T2DM, independently of their ability to induce weight loss. In a retrospective study, Colosimo et al. demonstrated a correlation between the glycated hemoglobin level, which was used to identify good glycemic responders, and the changes in FLI and FIB-4, independently of the body mass index (BMI) or the nature of the antidiabetic medication, represented by either GLP-1RAs, dipeptidyl peptidase-4 (DPP-4) inhibitors, or SGLT2 inhibitors [21].
Gameil et al. performed a study that compared the effect of liraglutide and dulaglutide vs. conventional treatment on FLI and FIB-4 index in patients with T2DM complicated with NAFLD. Liraglutide and dulaglutide led to significant improvements in the FLI and FIB-4 values, with a greater change in the liraglutide group [52].

Changes in Liver Fat Content or Fibrosis Evaluated by Imaging Techniques
Several studies investigated, using imaging techniques, the GLP-1RAs effect on the reduction of liver fat content as a primary outcome in patients with T2DM and NAFLD. The reduction of liver fat content was assessed by either ultrasound [45,56,59,69] or magneticresonance-based imaging methods [43,64,72]. Liraglutide improved the intrahepatic fat content, leading to a decrease of approximately 60% from baseline when compared with metformin and gliclazide, along with reducing mean HbA1c levels from 8.9% to 5.9% and transaminase levels [59]. Another study with liraglutide pointed to a close correlation between the degree of hepatic steatosis evaluated by ultrasound and the extent of weight loss [60]. Moreover, other authors suggest that the diminution of liver steatosis due to the significant decreases in body weight and HbA1c values can also lead to a reduction of the hepatic inflammation [83].
However, mixed results are reported in this category. Smits et al. did not observe any valuable effect after a 12-week liraglutide or sitagliptin administration in T2DM, where proton magnetic resonance spectroscopy ( 1 H-MRS) was used to measure hepatic fat and NFS, FIB-4 index, and AST to platelet ratio index (APRI) scores to assess fibrosis [62]. Another early study on T2DM patients randomized to insulin glargine or liraglutide for 12 weeks showed no significant change in liver proton density fat fraction measured by MRS [64], while dulaglutide was also not able to demonstrate a reduction of the intrahepatic fat estimated by transient elastography in NAFLD patients, probably due to the short duration of follow-up [84].
Contrariwise, Flint et al. found that semaglutide can determine more than 30% reduction in hepatic steatosis vs. placebo; however, no significant improvement of the liver stiffness assessed by magnetic resonance elastography (MRE) and of the magnetic resonance imaging-estimated proton density fat fraction (MRI-PDFF) was seen [70].
In short, the majority of studies acknowledge that GLP-1RAs decrease or even normalize the serum liver enzyme levels and reduce the liver fat content on imaging in people with NAFLD and T2DM [29,76].

Effects on Biopsy-Proven Histopathological Modifications
Liver biopsy and subsequent histologic examination represent the "gold standard" in diagnosing NAFLD [11]. There are only two important RCTs that have assessed the evolution of liver biopsy-proven histopathological abnormalities in diabetic and nondiabetic subjects with NASH [29,85]. The 48-week LEAN trial led by Armstrong and colleagues showed that liraglutide improved some features of NASH by delaying fibrosis progression when compared to placebo (39% vs. 9%, p = 0.019). However, the NAFLD Activity Score (NAS) did not display any significant changes during the trial [61]. The other RCT com-pared various doses of an atypical administration of once-daily subcutaneous semaglutide versus placebo for 72 weeks in patients with or without T2DM with histologic evidence of NASH. The study concluded that NASH resolution was dose-dependent, seen in 59% of those in the 0.4 mg/day semaglutide group (equivalent to 2.4 mg/week) versus 17% in the placebo group (p < 0.001) and due to the significant weight loss [71]. The treatment group exhibited a slower progression of fibrosis (4.9% vs. 18.8% in the placebo group), but no significant improvement in the severity of fibrosis [71]. Therefore, liraglutide and semaglutide achieved the histological resolution of NASH in 40 to 60% of participants, but with no influence on the overall fibrosis outcome [81,86]. The Effect of Semaglutide in Subjects with Noncirrhotic Nonalcoholic Steatohepatitis (ESSENCE) study is an ongoing phase 3 larger trial assessing the effects of semaglutide in NASH-related fibrosis [15,87].

Effects Assessed by Combined Investigations
Simeone et al. randomized patients with newly diagnosed T2DM and prediabetes to liraglutide 1.8 mg/dose or lifestyle counseling until achieving a target of approximately 7% less than the initial body weight. Both groups displayed significant comparable weight loss, improvement of the glycemic values, and reductions of the BMI, IL-1β level, and NAFLD degree assessed by magnetic resonance [54].
A 24-week, oral administration of semaglutide study assessed its efficacy and safety in T2DM complicated with NAFLD. A greater improvement was observed in the impaired liver function tests, hypertriglyceridemia, insulin resistance, and hepatic steatosis, led by the favorable effects on glycemic control and body weight. Controlled attenuation parameter (CAP) values significantly decreased from 344 to 279 dB/m at 24 weeks, the FIB-4 index decreased from 1.42 to 1.1, ferritin decreased from 4.1 ng/mL to 3.5 ng/mL, and hepatic enzymes were normalized at 24 weeks. However, liver stiffness measurement (LSM) values showed no significant changes in fibrosis after the 24 weeks of semaglutide treatment [67].
A systematic review and meta-analysis of 26 studies by Kumar et al. found that GLP-1RAs are associated with a significant reduction in ALT (mean difference (MD) −27.98, p = 0.04) and GGT (MD −40.65, p = 0.03), with no statistically significant influence upon AST values. They also demonstrated a significant improvement in liver steatosis (standard MD −2.53, p = 0.03), with insufficient data to support an effect on inflammation and fibrosis [88].
According to a meta-analysis of seven multinational RCTs assessing NAFLD by biopsy or imaging techniques, exenatide, and liraglutide proved to have consistent beneficial effects on serum liver enzymes, liver fat content, intraabdominal adipose tissue, and histological resolution of NASH [89].
At present, more large RCTs with biopsy-assessed outcomes are needed to accurately evaluate the effects of GLP-1RAs on various histopathological findings of NAFLD in patients with or without diabetes [77,83].

Indirect Effects That Promote NAFLD Improvement
The mechanisms involved in NAFLD pathophysiology are not completely understood. Current data link the development of fatty liver disease to insulin resistance, de novo lipogenesis, oxidative stress, microbiome dysregulation, immune or cytokine-related anomalies, mitochondrial injury, and apoptosis, overlapping a pre-existing genetic predisposition [7,90]. This multifactorial determinism suggests that NAFLD therapy should be as personalized as possible to optimally interfere with its predominant underlying mechanisms in each individual.
GLP-1RAs appear to have pleiotropic effects that can improve the metabolic liver disease in patients with T2DM [91]. Their effects on NAFLD may be due to systemic benefits upon various metabolic pathways, and not to the activation of the local GLP-1 receptors, which are not expressed in the hepatic tissue in significant amounts [92]. Among such metabolic effects, the GLP-1RA class mainly includes the stimulation of insulin release and the inhibition of glucagon secretion, a decreased hepatic glucose production, an increased insulin sensitivity in hepatocytes and adipocytes, a delayed gastric emptying, and the suppression of appetite [61,93]. This perspective is supported by the results of earlier studies, which indicated that an intensive glycemic control may ameliorate liver fibrosis seen on serial hepatic biopsies in patients with T2DM [94].
The loss of body weight by appetite suppression and inducing satiety is one of the main intermediate steps leading to the beneficial effects of GLP-1RAs. One example is the Lira-NAFLD study, where liraglutide 1.2 mg per day induced a mean weight loss of 3.6 kg and a proportional decrease of the liver fat content from 17.3% to 11.9% [60].
GLP-1RAs also have antioxidant and anti-inflammatory properties leading to a significant diminution of the oxidative stress and inflammatory biomarkers, which could determine NASH amelioration and some protection against the natural evolution of the liver fibrosis [95].
The gut microbiome plays an important role in the occurrence and development of NAFLD and T2DM, and can therefore emerge as a forthcoming target in the therapy of these metabolic diseases [96,97]. Animal studies provide some evidence that liraglutide has a beneficial impact on components of the intestinal flora that are related to inflammation and glucolipid metabolism, thus improving the fatty liver disease [98]. Liraglutide also seems to have a beneficial effect on the human intestinal microbiome in studies on patients with NAFLD, where it decreased the inflammatory factors and improved the liver function and the adipose content [99].

Direct Mechanisms That Promote NAFLD Improvement
Another pathway that warrants future investigation is the involvement of glycerophospholipid perturbance in T2DM pathogenesis [100]. Du et al. tried to use the changes in metabolites after treatment with two GLP-1RAs (liraglutide and dulaglutide) to evaluate the metabolic remodeling effects [101]. After assessing various metabolites in patients with T2DM, many studies pointed out that some of them contribute to specific metabolic dysfunctions, such as insulin resistance, obesity, glucose impairment, and NAFLD. At present, the most preeminent class of T2DM-associated metabolites is represented by glycerophospholipids. The authors of the abovementioned study point out that, for now, there is no clear understanding yet of how GLP-1RAs reduce major adverse cardiovascular events (MACE) or how they ameliorate NASH, as these effects are not fully explained by the indirect beneficial effects on weight, lipid profile, or blood pressure. They hypothesize that such effects may be determined by the effects liraglutide and dulaglutide have on different metabolic pathways, among which the alteration of glycerophospholipid metabolism may play an important role in the pathophysiology of NAFLD [101].
Another study investigated the effect of liraglutide on thyroid resistance in the liver of T2DM patients [102]. Relevant findings suggest that the intrahepatic impairment of the thyroid hormone (TH) pathway due to thyroid hormone receptor beta (THRB) gene mutations may lead to fatty liver; TH resistance can thus take part in NAFLD development [29]. In the abovementioned study, liraglutide decreased thyroid hormone levels in the NAFLD group of patients with T2DM. The results suggest that a mechanism by which liraglutide can improve hepatic TH resistance by restoring the impaired THRB expression is likely possible [102].

Perspectives
The twincretin tirzepatide is a novel dual glucagon-like peptide-1 (GLP-1) and glucosedependent insulinotropic polypeptide (GIP) receptor agonist approved by the FDA in May 2022 for the treatment of T2DM, based on the results of the Study of Tirzepatide in Participants with T2DM Not Controlled with Diet and Exercise Alone (SURPASS) clinical trials [103,104]; benefits in patients with obesity were also proved later [105]. A randomized trial of patients with T2DM who received tirzepatide revealed significant decreases in biomarkers related to NASH such as AST/ALT, procollagen III, keratin 18, and an increase in adiponectin levels, which is believed to activate antifibrotic and antisteatogenic effects in the liver [106]. A once-weekly subcutaneous administration of tirzepatide for 72 weeks in obese patients without diabetes demonstrated weight losses of 15% with the 5 mg dose, 19.5% with the 10 mg dose, and 20.9% with the 15 mg dose vs. a 3.1% weight loss in the placebo group. This RCT also showed changes in the fasting insulin levels, lipid profile, and blood pressure, along with a specific reduction of the fat mass [105]. This may be the primary indirect pathway leading to NAFLD improvement, but additional investigations are needed. Moreover, the prediabetes group that received tirzepatide had regressed to normoglycemia [105]. Given these beneficial effects in the metabolic profile and intrahepatic fat, an RCT with tirzepatide in biopsy-proven NASH individuals with or without T2DM is currently ongoing and will complete its results in 2024 [107].
Cotadutide is a dual GLP-1/glucagon (Gcg) receptor dual-agonist investigated at present for T2DM, kidney disease, heart failure, and NASH [108]. Cotadutide exerts a unique dual agonism mode of action upon the glucose and lipid metabolism that appears to be superior compared with GLP-1 agonism alone, given the indirect effects of reduced inflammation and reversed fibrosis found in preclinical models, in addition to significant properties to induce HbA1c and weight reduction and to improve insulin resistance [109]. In a 54-week phase 2b study, cotadutide led to positive changes in body weight, serum ALT levels, and scores of hepatic steatosis and fibrosis (NFS and FIB-4) in patients with NASH and T2DM [110].
Another relevant trial is the one-year administration of liraglutide to 82 women with NAFLD and history of gestational diabetes, followed by a major reduction of steatosis vs. placebo [111].
Last, but not least, it must be mentioned that multiple ongoing or forthcoming large trials are aiming to investigate, using liver biopsy or MRI techniques, the effects of GLP-1RAs and their newly derived molecules on patients with T2DM and NAFLD (Table 4). Their results will no doubt shape the clinical guidelines for pharmacological therapy in T2DM and NAFLD patients.
The increased appearance of NAFLD in patients with T2DM in a large proportion of patients, added to the still-incomplete knowledge of the mechanical changes that these therapies induce in the liver, supports the need for more studies that seek effective treatments for both pathologies. A new contingent of drugs under development based on the principle of GLP-1 receptor agonism may represent a turning point in this field in the coming years.   Abbreviations: T2DM, type 2 diabetes mellitus; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; GLP-1, glucagon-like peptide-1; NAS, NAFLD Activity Score; MRI-PDFF, magnetic resonance imaging-proton density fat fraction; oDI, Oral Disposition Index; OAD, oral antidiabetics; sc, subcutaneously; ALT, alanine aminotransferase; AST, aspartate aminotransferase; kPa, kilopascals; BMI, body mass index; IGT, impaired glucose tolerance.